Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1

ABSTRACT

The present invention concerns compositions and methods of use of anti-PD-1 antibodies comprising CDR sequences corresponding to SEQ ID NO:1 to SEQ ID NO:6. Preferably the antibody is a humanized antibody comprising the variable region amino acid sequences of SEQ ID NO: 9 and SEQ ID NO:10. The antibodies are of use to treat cancer and may be administered alone or with another standard anti-cancer therapy. The methods may comprise administering the anti-PD-1 antibody or antigen-binding fragment thereof in combination with one or more therapeutic agents such as antibody-drug conjugates, interferons (preferably interferon-α), and/or other checkpoint inhibitor antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/604,153, filed May 24, 2017, which claimed the benefit under35 U.S.C. 119(e) of provisional U.S. Patent Application 62/351,646,filed Jun. 17, 2016. The text of each priority application incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 24, 2017, isnamed IMM374US1 SL.txt and is 13,425 bytes in size.

FIELD

In certain embodiments, the present invention concerns compositions andmethods of use of novel antibodies against PD-1 (programmed cell deathprotein 1, aka CD279). Preferably, the anti-PD1 antibodies block bindingof PD-1 to its ligand, PD-L1. More preferably, the anti-PD1 antibody isa murine, chimeric or humanized antibody comprising the heavy chain CDRsequences GFAFSSNDMS (SEQ ID NO:1), TISGGGINTYYPDSVKG (SEQ ID NO:2) andRSNYAWFAY (SEQ ID NO:3) and the light chain CDR sequencesRASESVDTYGISFMN (SEQ ID NO:4), PNQGS (SEQ ID NO:5) and QQSKEVPWT (SEQ IDNO:6). Most preferably, the anti-PD1 antibody is a chimeric antibodycomprising the light chain variable region amino sequence of SEQ ID NO:7and the heavy chain variable region amino sequence of SEQ ID NO:8, or ahumanized antibody comprising the light chain variable region aminosequence of SEQ ID NO:9 and the heavy chain variable region aminosequence of SEQ ID NO:10. In certain preferred embodiments, theanti-PD-1 antibodies are of use to treat cancers that express PD-L1,although such methods are not limiting and various forms of cancer maybe treated with the subject antibodies, alone or in combination with oneor more other therapeutic agents. Exemplary cancers that may be treatedinclude, but are not limited to, non-small cell lung cancer (NSCLC),SCLC, mesothelioma, melanoma, breast cancer, ovarian cancer, coloncancer, prostate cancer, gastric cancer, renal-cell cancer, urothelialcancer, squamous cell carcinoma, head and neck cancer, non-Hodgkinlymphoma and Hodgkin lymphoma. The anti-PD-1 antibody may beadministered as a naked (unconjugated) antibody. In certain embodiments,the anti-PD-1 antibody may be administered in combination with at leastone other antibody or immunoconjugate, such as an anti-TAA(tumor-associated antigen) antibody or immunoconjugate. In alternativeembodiments, the anti-PD-1 antibody may be administered in combinationwith a T-cell redirecting bispecific antibody, such as ananti-CD19×anti-CD3, anti-Trop-2×anti-CD3, anti-CEACAM5×anti-CD3, or anyother known T-cell redirecting bsAb. In preferred embodiments, theanti-PD1 antibody may be administered in combination with an interferon,another checkpoint inhibitor antibody (e.g., anti-CTLA-4), anantibody-drug conjugate (ADC) or other anti-cancer therapy. Theanti-PD-1 therapy may exhibit enhanced or even synergistic activity whencombined with another therapeutic agent, and may be efficacious to treattumors that are resistant to or relapsed from standard cancer therapies.

BACKGROUND

A promising approach to immunotherapy concerns use of antagonisticantibodies against immune checkpoint proteins (e.g., Pardoll, 2012,Nature Reviews Cancer 12:252-64). Immune checkpoints function asendogenous inhibitory pathways for the immune system to maintainself-tolerance and to modulate the duration and extent of immuneresponse to antigenic stimulation (Pardoll, 2012). In their normalfunction, activity of checkpoint proteins modulates the immune responseto prevent development of autoimmune disease (e.g., He et al., 2017, JAutoimmun 79:1-3). However, it appears that tumor tissues may co-opt thecheckpoint system to reduce the effectiveness of the host immuneresponse, resulting in tumor growth (see, e.g., Pardoll, 2012, NatureReviews Cancer 12:252-64; Nirschl & Drake, 2013, Clin Cancer Res19:4917-24). Checkpoint molecules include CTLA-4 (cytotoxic T lymphocyteantigen-4), PD-1 (programmed cell death protein 1), PD-L1 (programmedcell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (Tcell immunoglobulin and mucin protein-3) and several others (Pardoll,2012, Nature Reviews Cancer 12:252-64; Nirschl & Drake, 2013, ClinCancer Res 19:4917-24).

Antibodies against several of the checkpoint proteins (CTLA-4, PD-1,PD-L1) are in clinical trials and have shown unexpected efficacy againsttumors that were resistant to standard treatments. Use in human cancertherapy has been approved by the FDAfor ipilimumab (anti-CTLA-4,Bristol-Myers Squibb) in malignant melanoma (Cameron et al, 2011, Drugs71:1093-104); pembrolizumab (anti-PD-1, Merck & Co.) in melanoma, headand neck cancer, Hodgkin lymphoma, urothelial cancer, gastric cancer andmetastatic NSCLC that expresses PD-1 (Press Release, Merck & Co., datedSep. 22, 2017, “FDA Approves Merck's KEYTRUDA® for Previously TreatedPatients with Recurrent Locally Advanced or Metastatic Gastric orGastroesophageal Junction Cancer Whose Tumors Express PD-1 (CPS GreaterThan or Equal to 1)”); nivolumab (anti-PD-1, Bristol-Myers Squibb) inmelanoma, lung cancer, kidney cancer, bladder cancer, head and neckcancer and Hodgkin lymphoma (Larkins et al., 2017, The Oncologist22:873-78; Kasamon et al., 2017, Oncologist 22:585-91), and fiveanti-PD-1 antibodies, for example, atezolizumab (anti-PD-L1, Roche) inbladder cancer and metastatic NSCLC (Ning et al., 2017, Oncologist22:743-49; Weinstock et al., 2017, Clin Cancer Res 23:4534-39).

Studies with checkpoint inhibitor antibodies for cancer therapy havegenerated unprecedented response rates in cancers previously thought tobe resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013,Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85; Pardoll, 2012, Nature Reviews 12:252-264; Mavilio & Lugli,2013, Oncoimmunology 2:e26535). Therapy with antagonistic checkpointblocking antibodies against CTLA-4, PD-1 and PD-1 is one of the mostpromising new avenues of immunotherapy for cancer and other diseases.However, a need continues to exist for more effective checkpointinhibitor antibodies, preferably with the ability to block binding ofPD-1 to PD-1.

SUMMARY

In certain embodiments, the present invention relates to murine,chimeric or humanized antibodies against PD-1, preferably comprising theheavy chain CDR sequences GFAFSSNDMS (SEQ ID NO:1), TISGGGINTYYPDSVKG(SEQ ID NO:2) and RSNYAWFAY (SEQ ID NO:3) and the light chain CDRsequences RASESVDTYGISFMN (SEQ ID NO:4), PNQGS (SEQ ID NO:5) andQQSKEVPWT (SEQ ID NO:6). Alternative embodiments relate to antibodiesthat bind to the same epitope as, or compete for binding to PD-1 with,an antibody having the amino acid sequences of SEQ ID NO:1 to SEQ IDNO:6. The antibody may be naked (unconjugated) or may be conjugated toone or more therapeutic or diagnostic agents, as discussed below. Otheralternative embodiments relate to antigen-binding fragments of thesubject anti-PD-1 antibodies, such as F(ab′)₂, F(ab)₂, F(ab′), F(ab), orscFv antibody fragments.

In preferred methods, the anti-PD-1 antibodies are of use to treatcancers, including but not limited to non-small cell lung cancer(NSCLC), SCLC, mesothelioma, melanoma, breast cancer, ovarian cancer,colon cancer, prostate cancer, gastric cancer, renal-cell cancer,urothelial cancer, squamous cell carcinoma, head and neck cancer,non-Hodgkin lymphoma and Hodgkin lymphoma. More preferably, the cancersto be to be treated express PD-1 and the subject methods may compriseassaying for PD-1 expression prior to therapy.

In certain embodiments, methods of use of anti-PD-1 antibodies orfragments thereof may involve combination therapy with other therapeutictreatments (Ott et al., 2017, J Immunother Cancer 5:16). Exemplarytreatments that may be used in combination therapy with the subjectanti-PD-1 antibodies or fragments thereof include use of interferons(e.g., interferon-α), other cytokines (e.g., GM-CSF, IL-2, IL-12, IL-15,IL-18, IL-21), other checkpoint inhibitor antibodies (e.g., anti-CTLA-4antibodies or anti-PD-1 antibodies), antibody-drug conjugates (ADCs,e.g., IMMU-132, IMMU-130, IMMU-140), radiation therapy, chemotherapy,talimogene laherparepvec (Chesney et al., J Clin Oncol, Oct. 5, 2017,[Epub ahead of print]), CAR-T cells (Rosewell et al., Sep. 14, 2017,[Epub ahead of print]), T-cell redirecting bispecific antibodies (Changet al., 2017, Cancer Res 77:5384-94), ibrutinib (Sagiv-Barfi et al.,2015, Proc Natl Acad Sci USA 112(9):E966-72), dacarbazine, paclitaxel,carboplatin, sargramostim (Wu et al., Int J Cancer, Aug. 22, 2017, [Epubahead of print]), selumetinib (Poon et a., 2017, J Immunother Cancer5:63), pemetrexed and cisplatin (Wehler et al., 2017, Lung Cancer108:212-26). These and other known anti-cancer therapies may be used incombination with checkpoint inhibitors to reduce tumor burden andenhance overall efficacy of treatment.

Certain embodiments concern combination with one or more additionalcheckpoint inhibitor antibodies. Such antibodies will be antagonisticfor checkpoint inhibitor function. Many such antibodies are known in theart, such as pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558,Bristol-Myers Squibb), pidilizumab (CT-011, CureTech Ltd.), AMP-224(Merck), MDX-1105 (Medarex), MEDI4736 (MedImmune), atezolizumab(MPDL3280A) (Genentech), BMS-936559 (Bristol-Myers Squibb), ipilimumab(Bristol-Myers Squibb), durvalumab (Astrazeneca) and tremelimumab(Pfizer). Anti-KIR antibodies such as lirlumab (Innate Pharma) andIPH2101 (Innate Pharma) may perform similar functions in NK cells.

In alternative embodiments, the subject anti-PD-1 antibodies orfragments thereof may be used in combination with an antibody-drugconjugate (ADC). ADCs are particularly effective for reducing tumorburden without significant systemic toxicity and may act to improve theeffectiveness of the immune response induced by checkpoint inhibitorantibodies. Exemplary ADCs approved for therapeutic use includegemtuzumab ozogamicin for AML (subsequently withdrawn from the market),brentuximab vedotin for ALCL and Hodgkin lymphoma, inotuzumab ozogamicinfor relapsed/refractory ALL and trastuzumab emtansine for HER2-positivemetastatic breast cancer (Verma et al., 2012, N Engl J Med 367:1783-91;Bross et al., 2001, Clin Cancer Res 7:1490-96; Francisco et al., 2003,Blood 102:1458-65; Lamb, Drugs, Aug. 17, 2017, [Epub ahead of print]).Numerous other candidate ADCs are currently in clinical testing, such asglembatumomab vedotin (Celldex Therapeutics), SAR3419 (Sanofi-Aventis),SAR56658 (Sanofi-Aventis), AMG-172 (Amgen), AMG-595 (Amgen), BAY-94-9343(Bayer), BIIB015 (Biogen Idec), BT062 (Biotest), SGN-75 (SeattleGenetics), SGN-CD19A (Seattle Genetics), vorsetuzumab mafodotin (SeattleGenetics), ABT-414 (AbbVie), ASG-5ME (Agensys), ASG-22ME (Agensys),ASG-16M8F (Agensys), IMGN-529 (ImmunoGen), IMGN-853 (ImmunoGen),MDX-1203 (Medarex), MLN-0264 (Millenium), RG-7450 (Roche/Genentech),RG-7458 (Roche/Genentech), RG-7593 (Roche/Genentech), RG-7596(Roche/Genentech), RG-7598 (Roche/Genentech), RG-7599 (Roche/Genentech),RG-7600 (Roche/Genentech), RG-7636 (Roche/Genentech), anti-PSMA ADC(Progenics), lorvotuzumab mertansine (ImmunoGen),milatuzumab-doxorubicin (Immunomedics), IMMU-130 (Immunomedics) andIMMU-132 (Immunomedics). (See, e.g., Li et al., 2013, Drug Disc Ther7:178-84; Firer & Gellerman, J Hematol Oncol 5:70; Beck et al., 2010,Discov Med 10:329-39; Mullard, 2013, Nature Rev Drug Discovery 12:329.)Preferably, where an ADC is used in combination with a checkpointinhibitor, the ADC is administered prior to the checkpoint inhibitor.

Tumor-associated antigens that may be targeted by ADCs of use incombination therapy include, but are not limited to, alpha-fetoprotein(AFP), a4 integrin, B7, carbonic anhydrase IX, complement factors C1q,C1r, C1s, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5a, C5aR, C5b, C5, C6,C7, C8, C9n, CCL19, CCL21, CD1, CD1a, CD2, CD3R, CD4, CDS, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD79b, CD80, CD83,CD86, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM-5, CEACAM-6, CSAp,DLL3, DLL4, ED-B, fibronectin, EGFR, EGP-1 (Trop-2), EGP-2, ErbB2,Factor H, FHL-1, fibrin, Flt-3, folate receptor, glycoprotein IIb/IIIa,gp41, gp120, GRO-β, HLA-DR, HM1.24, HM1.24, HMGB-1, hypoxia induciblefactor (HIF), Ia, ICAM-1, IFN-α, IFN-β, IFN-γ, IFN-λ, IgE, IGF-1R, IL-1,IL-1Ra, IL-2, IL-4R, IL-6, IL-6R, IL-8, IL-13R, IL-15R, IL-15, IL-17,IL-17R, IL-18, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,insulin-like growth factor-1 (ILGF-1), IP-10, KIR, Le(y),lipopolysaccharide (LPS), MAGE, MCP-1, mCRP, mesothelin, MIF, MIP-1A,MIP-1B, MUC1, MUC2, MUC3, MUC4, MUC5ac, NCA-90, NCA-95, NF-κB, P1GF,PSMA, RANTES, T101, TAC, TAG-72, tenascin, Thomson-Friedenreichantigens, thrombin, tissue factor, Tn antigen, TNF-α, TRAIL receptor (R1and R2), tumor necrosis antigens, VEGF, VEGFR and an oncogene product(see, e.g., Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani etal., J Immunol 2007, 178:1975-79; Novellino et al. Cancer ImmunolImmunother 2005, 54:187-207).

Exemplary antibodies that may be used for cancer therapy include, butare not limited to, hA19 (anti-CD19, U.S. Pat. No. 7,109,304), hR1(anti-IGF-1R, U.S. Pat. No. 9,441,043), hPAM4 (anti-MUC5ac, U.S. Pat.No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,151,164), hIMMU31(anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No.7,312,318), hLL2 (anti-CD22, U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAp,U.S. Pat. No. 7,387,772), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180),hMN-14 (anti-CEACAMS, U.S. Pat. No. 6,676,924), hMN-15 (anti-CEACAM6,U.S. Pat. No. 8,287,865), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785),hMN-3 (anti-CEACAM6, U.S. Pat. No. 7,541,440), Ab124 and Ab125(anti-CXCR4, U.S. Pat. No. 7,138,496), the Examples section of eachcited patent or application incorporated herein by reference.

The antibodies of use can be of various isotypes, preferably human IgG1,IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hinge andconstant region sequences. The antibodies or fragments thereof can bechimeric human-mouse, humanized (human framework and murinehypervariable (CDR) regions), or fully human, as well as variationsthereof, such as half-IgG4 antibodies (referred to as “unibodies”), asdescribed by van der Neut Kolfschoten et al. (Science 2007;317:1554-1557). More preferably, the antibodies or fragments thereof maybe designed or selected to comprise human constant region sequences thatbelong to specific allotypes, which may result in reduced immunogenicitywhen administered to a human subject. Preferred allotypes foradministration include a non-G1m1 allotype (nG1m1), such as G1m3,G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selectedfrom the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Combination therapy with immunostimulatory antibodies has been reportedto enhance efficacy, for example against tumor cells. Morales-Kastresanaet al. (2013, Clin Cancer Res 19:6151-62) showed that the combination ofanti-PD-1 (10B5) antibody with anti-CD137 (1D8) and anti-OX40 (OX86)antibodies provided enhanced efficacy in a transgenic mouse model ofhepatocellular carcinoma. Combination of anti-CTLA-4 and anti-PD-1antibodies has also been reported to be highly efficacious (Wolchok etal., 2013, N Engl J Med 369:122-33). Combination of rituximab withanti-KIR antibody, such as lirlumab (Innate Pharma) or IPH2101 (InnatePharma), was also more efficacious against hematopoietic tumors (Kohrtet al., 2012). The person of ordinary skill will realize that thesubject combination therapy may include combinations with multipleantibodies that are immunostimulatory and/or anti-tumor agents.

Alternative antibodies that may be used for treatment of various diseasestates include, but are not limited to, abciximab (anti-glycoproteinIIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab(anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20),panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20),trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab(anti-PD-1 receptor), ipilimumab (anti-CTLA-4), abagovomab(anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20),CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patent applicationSer. No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B(anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor),basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), atalizumab (anti-α4integrin), omalizumab (anti-IgE); anti-TNF-α antibodies such as CDP571(Ofei et al., 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI,M3TNFABI, M302B, M303 (Thermo Scientific, Rockford, Ill.), infliximab(Centocor, Malvern, Pa.), certolizumab pegol (UCB, Brussels, Belgium),anti-CD40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park,Ill.), BENLYSTA® (Human Genome Sciences); anti-CD38 antibodies such asMOR03087 (MorphoSys AG), MOR202 (Celgene), HuMax-CD38 (Genmab) ordaratumumab (Johnson & Johnson).

The subject checkpoint inhibitors may be administered in combinationwith one or more other immunomodulators to enhance the immune response.Immunomodulators may include, but are not limited to, a cytokine, achemokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), erythropoietin,thrombopoietin, tumor necrosis factor-α (TNF), TNF-β, granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, interferon-λ,stem cell growth factor designated “S1 factor”, human growth hormone,N-methionyl human growth hormone, bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin, NGF-β,platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I,insulin-like growth factor-II, macrophage-CSF (M-CSF), IL-1, IL-1α,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3,angiostatin, thrombospondin, endostatin, or lymphotoxin. In certainembodiments, an anti-PD-1 antibody or antibody fragment may be attachedto an immunomodulator, such as a cytokine. Cytokine complexes aredisclosed, for example, in U.S. Pat. Nos. 7,906,118 and 8,034,3522, theExamples section of each incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Binding of the murine parental 5G9.G1.B11 to PD-1 expressed onactivated Jurkat cells. Jurkat T cells (5 mL at 5×10⁵ cells/mL) were notstimulated or were stimulated with PHA (1 μg/mL), PMA (50 ng/mL), orboth PHA (1 μg/mL) and PMA (50 ng/mL) for 48 h and analyzed for theexpression of CD69 by flow cytometry. Expression of CD69 is a marker foractivated T cells. EH12 is a positive control for anti-PD-1

FIG. 2. Quantitation of IL-2 by ELISA. A notable increase in IL-2produced from T cells in a mixed lymphocyte assay was observed for5G9.G1.B11 dose-independently.

FIG. 3A. The amino acid sequence determined for the VK (SEQ ID NO:7) of5G9.G1.B11, with the 3 CDRs underlined.

FIG. 3B. The amino acid sequence determined for the VH (SEQ ID NO:8) of5G9.G1.B11, with the 3 CDRs underlined.

FIG. 3C. CDR sequences of 5G9.G1.B11 were, for the heavy chain,GFAFSSNDMS (SEQ ID NO:1), TISGGGINTYYPDSVKG (SEQ ID NO:2) and RSNYAWFAY(SEQ ID NO:3) and for the light chain, RASESVDTYGISFMN (SEQ ID NO:4),PNQGS (SEQ ID NO:5) and QQSKEVPWT (SEQ ID NO:6).

FIG. 4A. Binding of the chimeric 2G9 to recombinant PD-1-Fc by ELISA.

FIG. 4B. Binding of 2G9 to SpEFX-2D1, but not SpESX cells, by flowcytometry. The SpESX cell line, which does not express PD-1, wastransfected with human PD-1 to obtain SpESX-2D1, which overexpressesPD-1.

FIG. 5. Blockade of biotinlyated PD-1 binding to PD-1 on MDA-MB-231 by2G9.

FIG. 6. Amino acid sequence of light (SEQ ID NO:9) and heavy (SEQ IDNO:10) chains of humanized anti-PD-1 antibody (hPD-1). CDR sequences areunderlined. Framework residues (FRs) where the parental murine aminoacid residue is substituted with the corresponding human amino acidresidue are highlighted in bold font.

FIG. 7. DNA sequence encoding light chain (SEQ ID NO:11) and heavy chain(SEQ ID NO:12) of humanized anti-PD-1 antibody.

FIG. 8. Binding of humanized vs. chimeric anti-PD-1 to recombinant humanPD-1-His.

FIG. 9. Binding of humanized vs. chimeric anti-PD-1 to cells transfectedwith human PD-1 (2D1 cells).

FIG. 10. Combination therapy with anti-Trop-2×anti-CD3 bsAb andhumanized or chimeric anti-PD-1 antibody.

DETAILED DESCRIPTION

Definitions

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. An“antibody” includes monoclonal, polyclonal, bispecific, multispecific,murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, dAb and the like. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb).

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains, including human framework region(FR) sequences. The constant domains of the antibody molecule arederived from those of a human antibody. To maintain binding activity, alimited number of FR amino acid residues from the parent (e.g., murine)antibody may be substituted for the corresponding human FR residues.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A human antibodyalso can be constructed by genetic or chromosomal transfection methods,as well as phage display technology, all of which are known in the art.(See, e.g., McCafferty et al., 1990, Nature 348:552-553 for theproduction of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment. The fusion protein may comprisea single antibody component, a multivalent or multispecific combinationof different antibody components or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment and a therapeutic agent. Examples of therapeuticagents suitable for such fusion proteins include immunomodulators. Apreferred immunomodulator might be an interferon, such as interferon-α,interferon-β or interferon-λ.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity.

An antibody preparation, or a composition described herein, is said tobe administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient subject. In particular embodiments, anantibody preparation is physiologically significant if its presenceinvokes an antitumor response. A physiologically significant effectcould also be the evocation of a humoral and/or cellular immune responsein the recipient subject leading to growth inhibition or death of targetcells.

Checkpoint Inhibitors

In various embodiments, the subject anti-PD-1 antibody may beadministered in combination with one or more other checkpoint inhibitorantibodies. Various such antibodies are known and/or commerciallyavailable, primary targeted to PD-1, PD-1 or CTLA-4.

Programmed cell death protein 1 (PD-1, also known as CD279) encodes acell surface membrane protein of the immunoglobulin superfamily, whichis expressed in B cells and NK cells (Shinohara et al., 1995, Genomics23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45;Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews12:252-264). Anti-PD-1 antibodies have been used for treatment ofmelanoma, non-small-cell lung cancer, bladder cancer, prostate cancer,colorectal cancer, head and neck cancer, triple-negative breast cancer,leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N EnglJ Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Bergeret al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013,Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85).

Exemplary anti-PD-1 antibodies include lambrolizumab (MK-3475, MERCK),nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011,CURETECH LTD.). Anti-PD-1 antibodies are commercially available, forexample from ABCAM® (AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) andAFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).

A particular anti-PD-1 antibody of use, disclosed in the Examples below,is defined by the heavy chain CDR sequences GFAFSSNDMS (SEQ ID NO:1),TISGGGINTYYPDSVKG (SEQ ID NO:2) and RSNYAWFAY (SEQ ID NO:3) and thelight chain CDR sequences RASESVDTYGISFMN (SEQ ID NO:4), PNQGS (SEQ IDNO:5) and QQSKEVPWT (SEQ ID NO:6). The antibody may be used in chimeric,humanized, or fully human form, as discussed below.

Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is aligand for PD-1, found on activated T cells, B cells, myeloid cells andmacrophages. The complex of PD-1 and PD-1 inhibits proliferation ofCD8+T cells and reduces the immune response (Topalian et al., 2012, NEngl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).Anti-PDL1 antibodies have been used for treatment of non-small cell lungcancer, melanoma, colorectal cancer, renal-cell cancer, pancreaticcancer, gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013,Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51).

Exemplary anti-PDL1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PDL1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1).

Cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152) is also amember of the immunoglobulin superfamily that is expressed exclusivelyon T-cells. CTLA-4 acts to inhibit T cell activation and is reported toinhibit helper T cell activity and enhance regulatory T cellimmunosuppressive activity (Pardoll, 2012, Nature Reviews 12:252-264).Anti-CTL4A antibodies have been used in clinical trials for treatment ofmelanoma, prostate cancer, small cell lung cancer, non-small cell lungcancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al.,2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wadaet al., 2013, J Transl Med 11:89).

Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-MyersSquibb) and tremelimumab (PFIZER). Anti-PD-1 antibodies are commerciallyavailable, for example from ABCAM® (AB134090), SINO BIOLOGICAL INC.(11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572,PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recentlyreceived FDA approval for treatment of metastatic melanoma (Wada et al.,2013, J Transl Med 11:89).

These and other known checkpoint inhibitor antibodies may be used aloneor in combination other anti-cancer therapies as discussed herein. Theperson of ordinary skill will realize that methods of determiningoptimal dosages of checkpoint inhibitor antibodies to administer to apatient in need thereof, either alone or in combination with one or moreother agents, may be determined by standard dose-response and toxicitystudies that are well known in the art. In an exemplary embodiment, animmune checkpoint inhibitor antibody may preferably be administered atabout 0.3-10 mg/kg, or the maximum tolerated dose, administered aboutevery three weeks or about every six weeks. Alternatively, thecheckpoint inhibitor antibody may be administered by an escalatingdosage regimen including administering a first dosage at about 3 mg/kg,a second dosage at about 5 mg/kg, and a third dosage at about 9 mg/kg.Alternatively, the escalating dosage regimen includes administering afirst dosage of checkpoint inhibitor antibody at about 5 mg/kg and asecond dosage at about 9 mg/kg. Another stepwise escalating dosageregimen may include administering a first dosage of checkpoint inhibitorantibody about 3 mg/kg, a second dosage of about 3 mg/kg, a third dosageof about 5 mg/kg, a fourth dosage of about 5 mg/kg, and a fifth dosageof about 9 mg/kg. In another aspect, a stepwise escalating dosageregimen may include administering a first dosage of 5 mg/kg, a seconddosage of 5 mg/kg, and a third dosage of 9 mg/kg. Exemplary reporteddosages of checkpoint inhibitor mAbs include 3 mg/kg ipilimumabadministered every three weeks for four doses; 10 mg/kg ipilimumab everythree weeks for eight cycles; 10 mg/kg every three weeks for four cyclesthen every 12 weeks for a total of three years; 10 mg/kg MK-3475 everytwo or every three weeks; 2 mg/kg MK-3475 every three weeks; 15 mg/kgtremilimumab every three months; 0.1, 0.3, 1, 3 or 10 mg/kg nivolumabevery two weeks for up to 96 weeks; 0.3, 1, 3, or 10 mg/kg BMS-936559every two weeks for up to 96 weeks (Kyi & Postow, Oct. 23, 2013, FEBSLett [Epub ahead of print]; Callahan & Wolchok, 2013, J Leukoc Biol94:41-53).

Interferon Therapy

In other embodiments, the subject checkpoint inhibitors may beadministered in combination with interferon. Interferons are criticalrole players in the antitumor and antimicrobial host defense, and havebeen extensively explored as therapeutic agents for cancer (Billiau etal., 2006, Cytokine Growth Factor Rev 17:381-409; Pestka et al., 2004,Immunol Rev 202:8-32). Despite considerable efforts with type I and IIinterferons (IFN-α/β and γ), their use in clinic settings have beenlimited because of the short circulation half-life, systemic toxicity,and suboptimal responses in patients (Pestka et al., 2004, Immunol Rev202:8-32; Miller et al., 2009, Ann NY Acad Sci 1182:69-79). Thediscovery of the IFN-λ family in early 2003 brought an exciting newopportunity to develop alternative IFN agents for these unmet clinicalindications (Kotenko et al., 2003, Nat Immunol 4:69-77; Sheppard et al.,2003, Nat Immunol 4:63-8).

The therapeutic effectiveness of IFNs has been validated to date by theapproval of IFN-α2 for treating hairy cell leukemia, chronic myelogenousleukemia, malignant melanoma, follicular lymphoma, condylomataacuminata, AIDs-related Kaposi sarcoma, and chronic hepatitis B and C;IFN-β for treating multiple sclerosis; and IFN-γ for treating chronicgranulomatous disease and malignant osteopetrosis. Despite a vastliterature on this group of autocrine and paracrine cytokines, theirfunctions in health and disease are still being elucidated, includingmore effective and novel forms being introduced clinically (Pestka,2007, J. Biol. Chem 282:20047-51; Vilcek, 2006, Immunity 25:343-48). Theeffects of combination of various interferons with antibody-basedtherapies also remain under investigation.

In various embodiments, checkpoint inhibitor antibodies may be used incombination with one or more interferons, such as interferon-α,interferon-β or interferon-λ. Human interferons are well known in theart and the amino acid sequences of human interferons may be readilyobtained from public databases (e.g., GenBank Accession Nos. AAA52716.1;AAA52724; AAC41702.1; EAW56871.1; EAW56870.1; EAW56869.1). Humaninterferons may also be commercially obtained from a variety of vendors(e.g., Cell Signaling Technology, Inc., Danvers, Mass.; Genentech, SouthSan Francisco, Calif.; EMD Millipore, Billerica, Mass.).

Interferon-α (IFNα) has been reported to have anti-tumor activity inanimal models of cancer (Ferrantini et al., 1994, J Immunol 153:4604-15)and human cancer patients (Gutterman et al., 1980, Ann Intern Med93:399-406). IFNa can exert a variety of direct anti-tumor effects,including down-regulation of oncogenes, up-regulation of tumorsuppressors, enhancement of immune recognition via increased expressionof tumor surface MHC class I proteins, potentiation of apoptosis, andsensitization to chemotherapeutic agents (Gutterman et al., 1994, PNASUSA 91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20;Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int JOncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For sometumors, IFNα can have a direct and potent anti-proliferative effectthrough activation of STAT1 (Grimley et al., 1998 Blood 91:3017-27).Interferon-α2b has been conjugated to anti-tumor antibodies, such as thehL243 anti-HLA-DR antibody and depletes lymphoma and myeloma cells invitro and in vivo (Rossi et al., 2011, Blood 118:1877-84).

Indirectly, IFNα can inhibit angiogenesis (Sidky and Borden, 1987,Cancer Res 47:5155-61) and stimulate host immune cells, which may bevital to the overall antitumor response but has been largelyunder-appreciated (Belardelli et al., 1996, Immunol Today 17:369-72).IFNα has a pleiotropic influence on immune responses through effects onmyeloid cells (Raefsky et al, 1985, J Immunol 135:2507-12; Luft et al,1998, J Immunol 161:1947-53), T-cells (Carrero et al, 2006, J Exp Med203:933-40; Pilling et al., 1999, Eur J Immunol 29:1041-50), and B-cells(Le et al, 2001, Immunity 14:461-70). As an important modulator of theinnate immune system, IFNα induces the rapid differentiation andactivation of dendritic cells (Belardelli et al, 2004, Cancer Res64:6827-30; Paquette et al., 1998, J Leukoc Biol 64:358-67; Santini etal., 2000, J Exp Med 191:1777-88) and enhances the cytotoxicity,migration, cytokine production and antibody-dependent cellularcytotoxicity (ADCC) of NK cells (Biron et al., 1999, Ann Rev Immunol17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).

Interferon-β has been reported to be efficacious for therapy of avariety of solid tumors. Patients treated with 6 million units of IFN-βtwice a week for 36 months showed a decreased recurrence ofhepatocellular carcinoma after complete resection or ablation of theprimary tumor in patients with HCV-related liver cancer (Ikeda et al.,2000, Hepatology 32:228-32). Gene therapy with interferon-β inducedapoptosis of glioma, melanoma and renal cell carcinoma (Yoshida et al.,2004, Cancer Sci 95:858-65). Endogenous IFN-β has been observed toinhibit tumor growth by inhibiting angiogenesis in vivo (Jablonska etal., 2010, J Clin Invest. 120:1151-64.)

IFN-λs, designated as type III interferons, are a newly described groupof cytokines that consist of IFN-λ1, 2, 3 (also referred to asinterleukin-29, 28A, and 28B, respectively), that are geneticallyencoded by three different genes located on chromosome 19 (Kotenko etal., 2003, Nat Immunol 4:69-77; Sheppard et al., 2003, Nat Immunol4:63-8). IFN-λs activate signal transduction via the JAK/STAT pathwaysimilar to that induced by type I IFN, including the activation of JAK1and TYK2 kinases, the phosphorylation of STAT proteins, and theactivation of the transcription complex of IFN-stimulated gene factor 3(ISGF3) (Witte et al., 2010, Cytokine Growth Factor Rev 21:237-51; Zhouet al., 2007, J Virol 81:7749-58).

A major difference between type III and type I IFN systems is thedistribution of their respective receptor complexes. IFN-α/β signalsthrough two extensively expressed type I interferon receptors, and theresulting systemic toxicity associated with IFN-α/β administration haslimited their use as therapeutic agents (Pestka et al., 2007, J BiolChem 282:20047-51). In contrast, IFNαs signal through a heterodimericreceptor complex consisting of unique IFN-λ receptor 1 (IFN-λR1) andIL-10 receptor 2 (IL-10R2). As previously reported (Witte et al., 2009,Genes Immun 10:702-14), IFN-λR1 has a very restricted expression patternwith the highest levels in epithelial cells, melanocytes, andhepatocytes, and the lowest level in primary central nervous system(CNS) cells. Blood immune system cells express high levels of a shortIFN-λ receptor splice variant (sIFN-λR1) that inhibits IFN-λ action. Thelimited responsiveness of neuronal cells and immune cells implies thatthe severe toxicity frequently associated with IFN-α therapy may beabsent or significantly reduced with IFNλs (Witte et al., 2009, GenesImmun 10:702-14; Witte et al., 2010, Cytokine Growth Factor Rev21:237-51). A recent publication reported that while IFN-α and IFN-λinduce expression of a common set of ISGs (interferon-stimulated genes)in hepatocytes, unlike IFN-α, administration of IFN-λ did not induceSTAT activation or ISG expression in purified lymphocytes or monocytes(Dickensheets et al., 2013, J Leukoc Biol. 93, published online Dec. 20,2012). It was suggested that IFN-λ may be superior to IFN-α fortreatment of chronic HCV infection, as it is less likely to induceleukopenias that are often associated with IFN-α therapy (Dickensheetset al., 2013).

IFNλs display structural features similar to IL-10-related cytokines,but functionally possess type I IFN-like anti-viral andanti-proliferative activity (Witte et al., 2009, Genes Immun 10:702-14;Ank et al., 2006, J Virol 80:4501-9; Robek et al., 2005, J Virol79:3851-4). The anti-proliferative activity of IFNλs has beenestablished in several human cancer cell lines, including neuroendocrinecarcinoma BON1 (Zitzmann et al., 2006, Biochem Biophys Res Commun344:1334-41), glioblastoma LN319 (Meager et al., 2005, Cytokine31:109-18), immortalized keratinocyte HaCaT (Maher et al., 2008, CancerBiol Ther 7:1109-15), melanoma F01 (Guenterberg et al., 2010, Mol CancerTher 9:510-20), and esophageal carcinoma TE-11 (Li et al., 2010, Eur JCancer 46:180-90). In animal models, IFN-λs induce both tumor apoptosisand destruction through innate and adaptive immune responses, suggestingthat local delivery of IFN-λ might be a useful adjunctive strategy inthe treatment of human malignancies (Numasaki et al., 2007, J Immunol178:5086-98). A Fab-linked interferon-λ was demonstrated to have potentanti-tumor and anti-viral activity in targeted cells (Liu et al., 2013,PLoS One 8:e63940). In clinical settings, PEGylated IFN-λ1 (PEG-IFN-λ1)has been provisionally used for patients with chronic hepatitis C virusinfection. In a phase Ib study (n=56), antiviral activity was observedat all dose levels (0.5-3.0 μg/kg), and viral load reduced 2.3 to 4.0logs when PEG-IFN-λ1 was administrated to genotype 1 HCV patients whorelapsed after IFN-α therapy (Muir et al., 2010, Hepatology 52:822-32).At the same time, rates of adverse events commonly associated with typeI interferon treatment were lower with PEG-IFN-λ1 than with PEG-IFN-α.Neutropenia and thrombocytopenia were infrequently observed and therates of flu-like symptoms, anemia, and musculoskeletal symptomsdecreased to about ⅓ of that seen with PEG-IFN-α treatment. However,rates of serious adverse events, depression and other common adverseevents (≧10%) were similar between PEG-IFN-λ1 and PEG-IFN-α. Higherrates of hepatotoxicity were seen in the highest-dose PEG-IFN-λ1compared with PEG-IFN-α (“Investigational Compound PEG-Interferon LambdaAchieved Higher Response Rates with Fewer Flu-like and MusculoskeletalSymptoms and Cytopenias Than PEG-Interferon Alfa in Phase IIb Study of526 Treatment-Naive Hepatitis C Patients,” Apr. 2, 2011, Press Releasefrom Bristol-Myers Squibb).

In various embodiments, the subject checkpoint inhibitor mAbs may beused in combination with one or more interferons, such as interferon-α,interferon-β, interferon-λ1, interferon-λ2, or interferon-λ3. When usedwith other agents, the interferon may be administered prior to,concurrently with, or after the other agent. When administeredconcurrently, the interferon may be either conjugated to or separatefrom the other agent.

Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are a potent class of therapeuticconstructs that allow targeted delivery of cytotoxic agents to targetcells, such as cancer cells. Because of the targeting function, thesecompounds show a much higher therapeutic index compared to the samesystemically delivered agents. ADCs have been developed as intactantibodies or antibody fragments, such as scFvs. The antibody orfragment is linked to one or more copies of drug via a linker that isstable under physiological conditions, but that may be cleaved onceinside the target cell. ADCs approved for therapeutic use includegemtuzumab ozogamicin for AML (subsequently withdrawn from the market),inotuzumab ozogamicin for relapsed/refractory ALL, brentuximab vedotinfor ALCL and Hodgkin lymphoma, and trastuzumab emtansine forHER2-positive metastatic breast cancer (Verma et al., 2012, N Engl J Med367:1783-91; Bross et al., 2001, Clin Cancer Res 7:1490-96; Francisco etal., 2003, Blood 102:1458-65). Numerous other candidate ADCs arecurrently in clinical testing, such as glembatumomab vedotin (CelldexTherapeutics), SAR3419 (Sanofi-Aventis), SAR56658 (Sanofi-Aventis),AMG-172 (Amgen), AMG-595 (Amgen), BAY-94-9343 (Bayer), BIIB015 (BiogenIdec), BT062 (Biotest), SGN-75 (Seattle Genetics), SGN-CD19A (SeattleGenetics), vorsetuzumab mafodotin (Seattle Genetics), ABT-414 (AbbVie),ASG-5ME (Agensys), ASG-22ME (Agensys), ASG-16M8F (Agensys), IMGN-529(ImmunoGen), IMGN-853 (ImmunoGen), MDX-1203 (Medarex), MLN-0264(Millenium), RG-7450 (Roche/Genentech), RG-7458 (Roche/Genentech),RG-7593 (Roche/Genentech), RG-7596 (Roche/Genentech), RG-7598(Roche/Genentech), RG-7599 (Roche/Genentech), RG-7600 (Roche/Genentech),RG-7636 (Roche/Genentech), anti-PSMA ADC (Progenics), lorvotuzumabmertansine (ImmunoGen), milatuzumab-doxorubicin (Immunomedics), IMMU-130(Immunomedics), IMMU-132 (Immunomedics), IMMU-140 (Immunomedics) andantibody conjugates of pro-2-pyrrolinodoxorubicin. (See, e.g., Li etal., 2013, Drug Disc Ther 7:178-84; Firer & Gellerman, J Hematol Oncol5:70; Beck et al., 2010, Discov Med 10:329-39; Mullard, 2013, Nature RevDrug Discovery 12:329, U.S. Pat. No. 8,877,202.) Because of thepotential of ADCs to act as potent anti-cancer agents with reducedsystemic toxicity, they may be used either alone or as an adjuncttherapy to reduce tumor burden.

These and other known agents that stimulate immune response to tumorsand/or pathogens may be used in combination with checkpoint inhibitors.Other known co-stimulatory pathway modulators that may be used incombination include, but are not limited to, agatolimod, belatacept,blinatumomab, CD40 ligand, anti-B7-1 antibody, anti-B7-2 antibody,anti-B7-H4 antibody, AG4263, eritoran, anti-OX40 antibody, ISF-154, andSGN-70; B7-1, B7-2, ICAM-1, ICAM-2, ICAM-3, CD48, LFA-3, CD30 ligand,CD40 ligand, heat stable antigen, B7h, OX40 ligand, LIGHT, CD70 andCD24.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG_(i) constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: PHAGE DISPLAY LABORATORY MANUAL, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XENOMOUSE® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci. USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The V_(κ) sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the V_(κ) andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the V_(κ) andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, scFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692; U.S. Pat. No. 4,946,778; Raag andWhitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137(1991).

Techniques for producing single domain antibodies (DABs or VHH) are alsoknown in the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference. Singledomain antibodies may be obtained, for example, from camels, alpacas orllamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inacessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. 1 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:13) and veltuzumab (SEQ IDNO:14).

Rituximab heavy chain variable region sequence (SEQ ID NO: 13)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 14) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L  1 A — Veltuzumab G1m3R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nGlm 1 genotype, it is desirable to select the allotypeof the antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

Target Antigens and Exemplary Antibodies

In a preferred embodiment, combination therapy with anti-PD-1 mayinvolve antibodies that recognize and/or bind to antigens that areexpressed at high levels on target cells and that are expressedpredominantly or exclusively on diseased cells versus normal tissues.Exemplary antibodies of use for therapy of, for example, cancer includebut are not limited to LL1 (anti-CD74), LL2 or RFB4 (anti-CD22),veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab(GA101, anti-CD20), lambrolizumab (anti-PD-1), nivolumab(anti-PD-1),MK-3475 (anti-PD-1), AMP-224 (anti-PD-1), pidilizumab(anti-PD-1), MDX-1105 (anti-PD-L1), MEDI4736 (anti-PD-L1), MPDL3280A(anti-PD-L1), BMS-936559 (anti-PD-L1), ipilimumab (anti-CTLA-4),trevilizumab (anti-CTL4A), RS7 (anti-epithelial glycoprotein-1 (EGP-1,also known as Trop-2)), PAM4 or KC4 (both anti-mucin), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAMS),MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19),TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specificmembrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA),G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin), BWA-3(anti-histone H2A/H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1),PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), LG2-2 (anti-histoneH2B), and trastuzumab (anti-ErbB2).

Such antibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164),hA19 (U.S. Pat. No. 7,109,304), hIMIVIU-31 (U.S. Pat. No. 7,300,655),hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 5,789,554), hMu-9(U.S. Pat. No. 7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S.Pat. No. 6,676,924), hMN-15 (U.S. Pat. No. 8,287,865), hR1 (U.S. Pat.No. 9,441,043), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No.7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372,deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575) thetext of each recited patent or application is incorporated herein byreference with respect to the Figures and Examples sections.

Other useful antigens that may be targeted include alpha-fetoprotein(AFP), carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3,CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CEACAMS, CEACAM6, CTLA-4, DLL3, DLL4, VEGF(e.g., AVASTIN®, fibronectin splice variant), ED-B fibronectin (e.g.,L19), EGP-1 (Trop-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1) (e.g.,ERBITUX®), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor, Ga733,GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24, HER-2/neu,insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides,HCG, the HLA-DR antigen to which L243 binds, CD66 antigens, i.e.,CD66a-d or a combination thereof, MAGE, mCRP, MCP-1, mesothelin, MIP-1A,MIP-1B, macrophage migration-inhibitory factor (MIF), MUC1, MUC2, MUC3,MUC4, MUC5ac, placental growth factor (P1GF), PSA (prostate-specificantigen), PSMA, PAM4 antigen, PD-1 receptor, NCA-95, NCA-90, A3, A33,Ep-CAM, KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies forimmunotherapy, is Craig and Foon, Blood prepublished online Jan. 15,2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

Anti-cancer antibodies have been demonstrated to bind to histones insome case. Kato et al. (1991, Hum Antibodies Hybridomas 2:94-101)reported tha the lung cancer-specific human monoclonal antibody HB4C5binds to histone H2B. Garzelli et al. (1994, Immunol Lett 39:277-82)observed that Epstein-Barr virus-transformed human B lymphocytes producenatural antibodies to histones. In certain embodiments, antibodiesagainst histones may be of use in the subject combinations. Knownanti-histone antibodies include, but are not limited to, BWA-3(anti-histone H2A/H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1),PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2(anti-histone H2B) (see, e.g., Monestier et al., 1991, Eur J Immunol21:1725-31; Monestier et al., 1993, Molec Immunol 30:1069-75).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54). Milatuzumab (hLL1) is an exemplaryanti-CD74 antibody of therapeutic use for treatment of MIF-mediateddiseases.

An example of a most-preferred antibody/antigen pair is LL1, ananti-CD74 MAb (invariant chain, class II-specific chaperone, Ii) (see,e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; the Examples section of eachincorporated herein by reference). The CD74 antigen is highly expressedon B-cell lymphomas (including multiple myeloma) and leukemias, certainT-cell lymphomas, melanomas, colonic, lung, and renal cancers,glioblastomas, and certain other cancers (Ong et al., Immunology98:296-302 (1999)). A review of the use of CD74 antibodies in cancer iscontained in Stein et al., Clin Cancer Res. 2007 Sept. 15; 13(18 Pt2):55565-5563s, incorporated herein by reference. The diseases that arepreferably treated with anti-CD74 antibodies include, but are notlimited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung,renal, colonic cancers, glioblastome multiforme, histiocytomas, myeloidleukemias, and multiple myeloma.

The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Immunoconjugates

In certain embodiments, antibodies or fragments thereof of use incombination therapy may be conjugated to one or more therapeutic ordiagnostic agents. The therapeutic agents do not need to be the same butcan be different, e.g. a drug and a radioisotope. For example, ¹³¹I canbe incorporated into a tyrosine of an antibody or fusion protein and adrug attached to an epsilon amino group of a lysine residue. Therapeuticand diagnostic agents also can be attached, for example to reduced SHgroups and/or to carbohydrate side chains. Many methods for makingcovalent or non-covalent conjugates of therapeutic or diagnostic agentswith antibodies or fusion proteins are known in the art and any suchknown method may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br , for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MM. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F-Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

In certain preferred embodiments, the immunoconjugate may comprise acamptothecin drug, such as SN-38 (see, e.g., U.S. Pat. Nos. 7,999,083;8,999,344; 9,028,833). Camptothecin (CPT) and its derivatives are aclass of potent antitumor agents. Irinotecan (also referred to asCPT-11) and topotecan are CPT analogs that are approved cancertherapeutics (Iyer and Ratain, Cancer Chemother. Phamacol. 42: S31-S43(1998)). CPTs act by inhibiting topoisomerase I enzyme by stabilizingtopoisomerase I-DNA complex (Liu, et al. in The Camptothecins: UnfoldingTheir Anticancer Potential, Liehr J. G., Giovanella, B. C. andVerschraegen (eds), NY Acad Sci., NY 922:1-10 (2000)).

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 3 mg/kg and 20 mg/kg, more preferably between 4 mg/kg and 18mg/kg, more preferably between 6 mg/kg and 16 mg/kg, most preferablybetween 8 mg/kg and 12 mg/kg, preferably given either weekly, twiceweekly or every other week. The optimal dosing schedule may includetreatment cycles of two consecutive weeks of therapy followed by one,two, three or four weeks of rest, or alternating weeks of therapy andrest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. The dosage may be up to 24 mg/kg. Exemplary dosages of use mayinclude 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24mg/kg. Preferred dosages are 4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg. Theperson of ordinary skill will realize that a variety of factors, such asage, general health, specific organ function or weight, as well aseffects of prior therapy on specific organ systems (e.g., bone marrow)may be considered in selecting an optimal dosage of immunoconjugate, andthat the dosage and/or frequency of administration may be increased ordecreased during the course of therapy. The dosage may be repeated asneeded, with evidence of tumor shrinkage observed after as few as 4 to 8doses. The optimized dosages and schedules of administration disclosedherein show unexpected superior efficacy and reduced toxicity in humansubjects, which could not have been predicted from animal model studies.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including the parental compound, CPT-11, from which SN-38 isderived in vivo.

Therapeutic Agents

In certain alternative embodiments involving combination therapy,therapeutic agents may be administered as conjugates (for example, ADCs)or in unconjugated form. Exemplary therapeutic agents of use includecytotoxic agents, anti-angiogenic agents, pro-apoptotic agents,antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs,toxins, enzymes or other agents. Drugs of use may possess apharmaceutical property selected from the group consisting ofantimitotic, antikinase, alkylating, antimetabolite, antibiotic,alkaloid, anti-angiogenic, pro-apoptotic agents and combinations thereof

Exemplary drugs of use may include, but are not limited to,5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogenreceptor binding agents, etoposide (VP16), etoposide glucuronide,etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -λ., and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT.

Radionuclides of use include, but are not limited to- ¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, and ²²⁷Th. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹C,¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru,¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm,¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹b, and the like.Some useful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or, ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983),130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bc1-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA. The skilledartisan will realize that any siRNA or interference RNA species may beattached to an antibody or fragment thereof for delivery to a targetedtissue. Many siRNA species against a wide variety of targets are knownin the art, and any such known siRNA may be utilized in the claimedmethods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bc12 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats. Methods of cancer treatment may involve use of anti-PD-1antibodies alone, or in combination with one or more other therapeuticmodalities.

The administration of anti-PD-1 can be supplemented by administeringconcurrently or sequentially a therapeutically effective amount ofanother antibody that binds to or is reactive with an antigen on thesurface of the target cell, such as a tumor-associated antigen (TAA).Preferred additional MAbs comprise at least one humanized, chimeric orhuman MAb selected from the group consisting of a MAb reactive withalpha-fetoprotein (AFP), a4 integrin, B7, carbonic anhydrase IX,complement factors C1q, C1r, C1s, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b,C5a, C5aR, C5b, C5, C6, C7, C8, C9n, CCL19, CCL21, CD1, CD1a, CD2, CD3R,CD4, CDS, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22,CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44,CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74,CD79a, CD79b, CD80, CD83, CD86, CD95, CD126, CD133, CD138, CD147, CD154,CEACAM-5, CEACAM-6, CSAp, DLL3, DLL4, ED-B, fibronectin, EGFR, EGP-1(Trop-2), EGP-2, ErbB2, Factor H, FHL-1, fibrin, Flt-3, folate receptor,glycoprotein IIb/IIIa, gp41, gp120, GRO-β, HLA-DR, HM1.24, HM1.24,HMGB-1, hypoxia inducible factor (HIF), Ia, ICAM-1, IFN-α, IFN-β, IFN-γ,IFN-λ, IgE, IGF-1R, IL-1, IL-1Ra, IL-2, IL-4R, IL-6, IL-6R, IL-8,IL-13R, IL-15R, IL-15, IL-17, IL-17R, IL-18, IL-18R, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-1 (ILGF-1),IP-10, KIR, Le(y), lipopolysaccharide (LPS), MAGE, MCP-1, mCRP,mesothelin, MIF, MIP-1A, MIP-1B, MUC1, MUC2, MUC3, MUC4, MUC5ac, NCA-90,NCA-95, NF-κB, P1GF, PSMA, RANTES, T101, TAC, TAG-72, tenascin,Thomson-Friedenreich antigens, thrombin, tissue factor, Tn antigen,TNF-α, TRAIL receptor (R1 and R2), tumor necrosis antigens, VEGF, VEGFRor an oncogene product. Various antibodies of use, such as anti-CD19,anti-CD20, and anti-CD22 antibodies, are known to those of skill in theart. See, for example, Ghetie et al., Cancer Res. 48:2610 (1988); Hekmanet al., Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. Opin.Oncol. 8:353 (1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393;6,676,924; 7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785;7,238,786; 7,282,567; 7,300,655; 7,312,318; 7,501,498; 7,612,180;7,670,804; and U.S. Patent Application Publ. Nos. 20080131363;20070172920; 20060193865; and 20080138333, the Examples section of eachincorporated herein by reference.

The combination therapy can be further supplemented with theadministration, either concurrently or sequentially, of at least onetherapeutic agent. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1.

The combinations of therapeutic agents can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe checkpoint inhibitor antibody is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The subject anti-PD-1 antibodies can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, the antibody is infused over a period of less than about 4hours, and more preferably, over a period of less than about 3 hours.For example, the first bolus could be infused within 30 minutes,preferably even 15 min, and the remainder infused over the next 2-3 hrs.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic combinations. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the agents to be administered. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release from such a matrix depends upon the molecular weight of thetherapeutic agent, the amount of agent within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. 1 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The checkpoint inhibitor antibodies may be administered to a mammalsubcutaneously or even by other parenteral routes, such asintravenously, intramuscularly, intraperitoneally or intravascularly.ADCs may be administered intravenously, intraperitoneally orintravascularly. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. Preferably, the checkpointinhibitor antibody is infused over a period of less than about 4 hours,and more preferably, over a period of less than about 3 hours.

More generally, the dosage of an administered checkpoint inhibitorantibody for humans will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history and treatments. It may be desirable to providethe recipient with a dosage of anti-PD-1 antibody that is in the rangeof from about 1 mg/kg to 25 mg/kg as a single intravenous infusion,although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy.

Alternatively, a checkpoint inhibitor antibody may be administered asone dosage every 2 or 3 weeks, repeated for a total of at least 3dosages. Or, a combination may be administered twice per week for 4-6weeks. If the dosage is lowered to approximately 200-300 mg/m² (340 mgper dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), itmay be administered once or even twice weekly for 4 to 10 weeks.Alternatively, the dosage schedule may be decreased, namely every 2 or 3weeks for 2-3 months. It has been determined, however, that even higherdoses, such as 20 mg/kg once weekly or once every 2-3 weeks can beadministered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

The person of ordinary skill will realize that while the dosageschedules discussed above are relevant for anti-PD-1, the interferonagents should be administered at substantially lower dosages to avoidsystemic toxicity. Dosages of interferons (such as PEGINTERFERON) forhumans are more typically in the microgram range, for example 180 μgs.c. once per week, or 100 to 180 μg, or 135 μg, or 135 μg/1.73 m², or90 μg/1.73 m², or 250 μg s.c. every other day may be of use, dependingon the type of interferon.

While the checkpoint inhibitor antibodies may be administered as aperiodic bolus injection, in alternative embodiments the checkpointinhibitor antibodies may be administered by continuous infusion. Inorder to increase the Cmax and extend the PK of the therapeutic agentsin the blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, the checkpoint inhibitor antibodies can be administered byintravenous infusions over relatively short periods of 2 to 5 hours,more preferably 2-3 hours.

In preferred embodiments, the anti-PD-1 and/or combination is of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising and spreading as metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor). Cancers conducive to treatmentmethods of the present invention involves cells which express,over-express, or abnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational TROPhoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BASICPATHOLOGY, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an anti-PD-1 antibody, antibody fragment, or fusionprotein. Various embodiments relate to expression vectors comprising thecoding DNA sequences. The vectors may contain sequences encoding thelight and heavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327, 7,537,930, 7,785,880, 8,076,410, 8,153,433 and8,372,603, the Examples section of each incorporated herein byreference.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more checkpoint inhibitor antibodies and/or othertherapeutic agents as described herein. If the composition containingcomponents for administration is not formulated for delivery via thealimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1 Generation and Use of Chimeric anti-PD-1 Antibody (IMMU-cPD-1)

The blocking (antagonistic) anti-PD-1 monoclonal antibody 5G9.G1.B11 andits chimeric counterpart 2G9 (IMMU-cPD-1) were generated as follows.BALB/c mice were immunized with recombinant human PD-1-Fc fusion protein(AB Biosciences), resulting in the isolation of a positive clone (5G9)by hybridoma technology. To ensure monoclonality, 5G9 was subclonedtwice, yielding 5G9.G1.B11 (5G9 for short). The 5G9 mAb was purified tohomogeneity, as shown by SE-HPLC and SDS-PAGE analyses (data not shown).The reactivity of 5G9 for PD-1 was confirmed by SE-HPLC with its bindingto recombinant PD-1-His (not shown), and by flow cytometry with itsbinding to PD-1-expressed on activated Jurkat T cells (FIG. 1).Importantly, the blocking activity of 5G9 was demonstrated by adose-dependent, notable increase of IL-2 secreted by T cells in a mixedlymphocyte assay (FIG. 2). The amino acid sequences pertaining to theV_(K) and V_(H) of 5G9 were provided in FIG. 3A and FIG. 3B,respectively. The CDRs of 5G9 are further delineated in FIG. 3C.

A chimeric version of 5G9, comprising the V_(H) and V_(K) of 5G9 andhuman Fc of IgG1, was generated, and the mAb from the lead clone (2G9)was purified and shown to be homogeneous by SDS-PAGE (not shown). Thebinding of 2G9 to recombinant PD-1-Fc with a high affinity of 70 pM wasdemonstrated by ELISA (FIG. 4A), and further confirmed by flow cytometry(FIG. 4B) using a subline of SpESFX transfected to overexpress PD-1(SpESFX-2D1). The blocking activity of 2G9 was similar to that of 5G9 orEH12, as demonstrated by inhibiting the binding of biotinylated PD-1 tothe endogenous PD-1 expressed on MDA-MB-231 (FIG. 5).

Example 2 Humanized Anti-PD-1 Antibody

A humanized anti-PD-1 antibody was constructed as follows. The V genesof the anti-PD-1 antibody from Example 1 above were identified by PCRamplification and DNA sequencing. cDNA was prepared from a transfectedcell line producing a murine anti-PD-1 antibody by general molecularcloning techniques (Sambrook et al., Molecular Cloning, A laboratorymanual, 2nd Ed (1989)). The Vk sequence for the MAb was amplified usingthe extended primer set described by Leung et al. (BioTechniques, 15:286 (1993)). The V_(H) sequence was amplified using the primersannealing to the constant region of murine IgG described by Leung et al.(Hybridoma, 13:469 (1994)).

PCR reaction mixtures containing 10 μl of the first strand cDNA product,10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),250 μM of each dNTP, 200 nM of the primers, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus) were subjected to 30 cycles of PCR. EachPCR cycle consisted of denaturation at 94° C. for 1 min, annealing at50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min. AmplifiedVk and V_(H) fragments were purified on 2% agarose (BioRad, Richmond,Calif.).

To confirm their authenticity, the cloned Vk and V_(H) genes wereexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)) and shown inExample 1. The sequences of the Vk and V_(H) variable regions weredetermined by Sanger dideoxy sequencing.

Based on the V gene sequences, a humanized anti-PD-1 antibody wasdesigned and constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)). PCR products for Vk were subclonedinto a staging vector that contains an Ig promoter, a signal peptidesequence and convenient restriction sites to facilitate in-frameligation of the Vk. PCR products. PCR products for V_(H) were subclonedinto a similar staging vector. Individual clones containing therespective PCR products were sequenced by the method of Sanger et al.(Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)).

For both V_(H) and V_(κ) chains, the amino acid sequences of thehumanized anti-hPD-1 antibody, designated hPD-1-v5, were constructed asfollows. The CDR sequences of the murine or chimeric anti-PD-1 wereretained. The amino acid residues of the murine frameworks were eachqueried against the cognate one in the human germline data base; if aresidue was the same as the one in the human framework, it was retained;otherwise, it was replaced with a human residue found with the highestfrequency at that position. Finally, amino acid residues adjacent to theCDRs were generally retained. A series of humanized PD-1 antibodies wereprepared as discussed above. The version 4 (V4) antibody contained aputative N-glycosylation site on the light chain, which was removed toproduce the version 5 (V5) antibody.

FIG. 6 shows the resulting amino acid sequences of the light (SEQ IDNO:9) and heavy (SEQ ID NO:10) chains of hPD-1-v5. The CDR sequences areunderlined. Framework region (FR) residues where the murine FR residuewas replaced with the corresponding human residue from the germline areindicated in bold. For the light chain the substitutions wereconcentrated in FR3, while for the heavy chain they were distributedthroughout the humanized antibody. The corresponding DNA sequencesencoding the light (SEQ ID NO:11) and heavy (SEQ ID NO:12) chains ofhPD-1-v5 are shown in FIG. 7. They were synthesized, assembled withtheir respective human kappa and human IgG1 constant domains into apdHL2-based vector, and expressed in SpESF-X10 to obtain hPD-1-v5.

Vk and V_(H) expression cassettes were assembled in the modified stagingvectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/HindIIIfragments, respectively, and subcloned into a single expression vector,pdHL2, as described by Gilles et al. (J. Immunol. Methods 125:191 (1989)and also shown in Losman et al., Cancer, 80:2660 (1997)).

Co-transfection and assay for antibody secreting clones by ELISA werecarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) were usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells were grown in 96-wellmicrotiter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process wasinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerged 2-3 weekspost-electroporation. The cultures were then expanded for furtheranalysis. Transfectoma clones that are positive for the secretion ofchimeric, humanized or human heavy chain were identified by ELISA assay.

Protein A-purified hPD-1-v5 and an earlier version (hPD-1-v4), whichcontained a putative N-glycosylation site in the V_(K) FR3, werecompared to chimeric anti-PD1 in vitro, by binding to recombinant humanPD-1-His. As shown in FIG. 8, both humanized anti-hPD-1 antibodiesexhibited somewhat lower affinities for recombinant PD-1-His, with EC₅₀values of 148 and 190 pM vs. 43 pM for cPD-1. The slightly loweraffinity of hPD-1-v5 for the target antigen was also observed in 2D1cells transfected with human PD-1 antigen, with EC₅₀ values of 0.168μg/mL (1.1 nM) for cPD1 vs. 0.568 μg/mL (3.8 nM) for hPD-1-v5 (FIG. 9).

Significantly, as described in Example 3 below, both cPD-1 and hPD-1-v5enhanced the efficacy of an (E1)-3s bsAb, targeting Trop-2 and CD3.Anti-tumor activity was assessed by MTS assay against MDA-MB-231 humanbreast cancer cells. In the case of cPD1, the anti-hPD-1 antibodyresulted in a decrease in EC₅₀ from 4.7 pM with (E1)-35 alone to 1.1 pMwith (E1)-3s plus cPD-1. For the humanized antibody, the addition ofhPD-1-v5 resulted in a decrease from 4.7 pM to 2.1 pM, i.e. over atwo-fold decrease in EC₅₀. These results shown that the chimeric andhumanized anti-hPD1 antibody is capable of enhancing the anti-tumorefficacy of T-cell redirecting bsAbs.

Example 3 Combination Therapy with T-Cell Redirecting bsAb and Anti-PD-1Checkpoint Inhibitor Antibody

Bispecific antibodies (bsAbs) for redirecting T cells to cancers haveshown promise in both pre-clinical and clinical studies. However,clinical success has been minimal for solid cancers to date. Previously,we reported highly effective T-cell redirected therapy of pancreatic andgastric tumors in xenograft models using a trivalent bsAb, designated(E1)-3s, which comprises an anti-CD3 scFv covalently conjugated to astabilized dimer of a Trop-2-targeting Fab (Rossi et al., 2014, MolCancer Ther 13:2341-51). Trop-2 is highly expressed in diverseepithelial cancers, including breast, lung, gastric, colorectal,pancreatic, bladder, ovarian, uterine and prostate carcinomas, withlimited presence on normal human tissues. Compared to first generationbsAbs (e.g. BiTE), which induce high-level cytokine production that canlead to serious side effects, efficient T-cell killing is mediated by(E1)-35 with minimal cytokine release (Rossi et al., 2014).

Herein, we report the potential utility of (E1)-3s for therapy of breastcancers, including TNBC. Additionally, we show for the first time thataddition of a checkpoint inhibitor can enhance bsAb-redirected T-celltherapy. IMMU-cPD-1 is a chimeric mAb that binds with high affinity tohuman PD-1 and efficiently blocks binding to its ligand, PD-1 . TheMDA-MB-231 human TNBC cell line has relatively low levels of surfaceTrop-2 (36,000/cell) and expresses PD-1 constitutively. In ex-vivoassays, where MDA-MB-231 cells were mixed with purified human T cells,(E1)-3s mediated potent T-cell redirected killing (IC₅₀<10 pM). Additionof IMMU-cPD-1 enhanced (E1)-3s-mediated T-cell killing.

The advantage of combining the checkpoint inhibitor with redirectedT-cell therapy was supported with in-vivo xenograft studies. NOD-SCIDmice were co-injected with purified human T cells (2.5×10⁶) andMDA-MB-231 (5×10⁶). Groups were administered: 1) five daily injectionsof (E1)-3s; 2) cPD-1 twice weekly for 4 weeks; or 3) a combination of(E1)-3s and cPD-1 treatments. An untreated control group, comprising Tcells and tumor cells, reached the endpoint (tumors >1 cm³) on day 35,at which point mice treated with (E1)-35 had smaller tumors (P=0.0023,AUC). Treatment with (E1)-3s improved median survival to 42 days(P=0.0019, log-rank).The group treated with the combination of (E1)-3sand IMMU-cPD-1 had significantly smaller tumor volumes (P=0.0121, AUC,Day 42) and longer median survival (49 days, P=0.008, log-rank),compared to those treated with (E1)-3s alone. Treatment with IMMU-cPD-1alone did not retard tumor growth or improve survival, compared to theuntreated control group. In conclusion, (E1)-3s is an attractivecandidate for T-cell redirected therapy of breast cancer due to itspotent activity with potentially reduced side effects and the prevalenceof Trop-2 expression associated with this disease. Tumor micro arraysrepresenting 117 breast cancer patients showed >85% positivity forTrop-2. Our immunohistochemical analysis of more than 50 individual TNBCpatient specimens demonstrated 92% positivity with 80% having moderateto strong Trop-2 staining. Combining checkpoint inhibitors withredirected T cell therapy may represent a new paradigm for themanagement of solid cancers, including breast cancer.

Example 4 ADC Therapy with IMMU-132 for Metastatic Solid Cancers

IMMU-132 is an ADC comprising the active metabolite of CPT-11, SN-38,conjugated by a pH-sensitive linker (average drug-antibody ratio=7.6) tothe hRS7 anti-Trop-2 humanized monoclonal antibody, which exhibits rapidinternalization when bound to Trop-2. IMMU-132 targets Trop-2, a type Itransmembrane protein expressed in high prevalence and specificity bymany carcinomas. This Example reports a Phase I clinical trial of 25patients with different metastatic cancers (pancreatic, 7;triple-negative breast [TNBC], 4; colorectal [CRC], 3; gastric, 3,esophageal, prostatic, ovarian, non-small-cell lung, small-cell lung[SCLC], renal, tonsillar, urinary bladder, 1 each) after failing amedian of 3 prior treatments (some including topoisomerase-I and -IIinhibiting drugs).

IMMU-132 was administered in repeated 21-day cycles, with each treatmentgiven on days 1 and 8. Dosing started at 8 mg/kg/dose (i.e., 16mg/kg/cycle), and escalated to 18 mg/kg before encounteringdose-limiting neutropenia, in a 3+3 trial design. Fatigue, alopecia, andoccasional mild to moderate diarrhea were some of the more commonnon-hematological toxicities, with 2 patients also reporting a rash.Over 80% of 24 assessable patients had stable disease or tumor shrinkage(SD and PR) among the various metastatic cancers as best response by CT.Three patients (CRC, TNBC, SCLC) have PRs by RECIST 1.1; median TTP forall patients, excluding those with pancreatic cancer, is >18 weeks.Neutropenia has been controlled by dose reduction to 8-10 mg/kg/dose(16-20 mg/kg/cycle).

Immunohistochemistry showed strong expression of Trop-2 in most archivedpatient tumors, but it is not detected in serum. Correspondingreductions in blood tumor marker titers (e.g., CEA, CA19-9) reflectedtumor responses. No anti-antibody or anti-SN-38 antibodies have beendetected despite repeated dosing. Peak and trough assessments ofIMMU-132 concentrations in the serum show that the conjugate clearscompletely within 7 days, an expected finding based on in vitro studiesshowing 50% of the SN-38 is released in the serum every day. Theseresults indicate that this novel ADC, given in doses ranging from 16-24mg/kg per cycle, shows a high therapeutic index in diverse metastaticsolid cancers.

Example 5 IMMU-130, an SN-38 ADC That Targets CEACAMS, isTherapeutically Active in Metastatic Colorectal Cancer (mCRC)

IMMU-130, an ADC of SN-38 conjugated by a pH-sensitive linker (7.6average drug-antibody ratio) to the humanized anti-CEACAMS antibody(labetuzumab), is completing two Phase I trials. In both, eligiblepatients with advanced mCRC were required to have failed/relapsedstandard treatments, one being the topoisomerase-I inhibiting drug,CPT-11 (irinotecan), and an elevated plasma CEA (>5 ng/mL).

IMMU-130 was administered every 14 days (EOW) at doses starting from 2.0mg/kg in the first protocol (IMMU-130-01). Febrile neutropenia occurredin 2 of 3 patients at 24 mg/kg; otherwise at ≦16 mg/kg, neutropenia(≧Grade 2) was observed in 7 patients, with one also experiencingthrombocytopenia. One patient [of 8 who received ≧4 doses (2 cycles)]showed a 40.6% decrease in liver (starting at 7 cm) and lung targetlesions (PR by RECIST) for 4.7 months, with no major toxicity,tolerating a total of 18 doses at 16 mg/kg. The study continues at 12mg/kg EOW.

Since SN-38 is most effective in S-phase cells, a more protractedexposure could improve efficacy. Thus, in a second Phase I trial(IMMU-130-02), dosing was intensified to twice-weekly, starting at 6mg/kg/dose for 2 weeks (4 doses) with 1 week off, as a treatment cycle,in a 3+3 trial design. Neutropenia and manageable diarrhea were themajor side effects, until dose reduction to 4.0 mg/kg twice-weekly, withearly results indicating multiple cycles are well-tolerated. Currently,tumor shrinkage occurred in 3 patients, with 1 in continuing PR (-46%)by RECIST, among 6 patients who completed ≧4 doses (1 cycle). In bothtrials, CEA blood titers correlated with tumor response, and high levelsdid not interfere with therapy. There have been no anti-antibody oranti-SN-38 antibody reactions, based on ELISA tests. In each study, theADC was cleared by 50% within the first 24 h, which is much longerexposure than with typical doses of the parental molecule, CPT-11. Theseresults indicate that this novel ADC, given in different regimensaveraging ˜16-24 mg/kg/cycle, shows a high therapeutic index in advancedmCRC patients. Since CEACAM5 has elevated expression in breast and lungcancers, as well as other epithelial tumors, it may be a useful targetin other cancers as well.

Example 6 Antitumor Activity of Checkpoint Inhibitor Antibody Alone orCombined with T-Cell Redirecting bsAb, IFN-α or ADC

To determine if the antitumor activity of an exemplary checkpointinhibitor antibody, hPD-1-v5 (anti-PD-1), is synergistic with orinhibited by the addition of other therapeutic agents, hPD-1-v5 mAb isevaluated alone or in combination with the exemplary T-cell redirectingbsAb (E1)-3s, with interferon-α (PEGINTERFERON®), or with the exemplaryADC hRS7-SN-38 (IMMU-132) in murine tumor models. M109 lung carcinoma,SA1N fibrosarcoma, and CT26 colon carcinoma models are chosen based ondifferent sensitivity to the various agents and PD-1 blockade. Human Tcells are co-administered with the antibodies.

All compounds are tested at their optimal dose and schedule. When usedin combination, hPD-1-v5 mAb is initiated one day after the first doseof IMMU-132, (E1)-3s or interferon-α. Percent tumor growth inhibitionand number of days to reach target tumor size are used to evaluateefficacy. Antitumor activity is scored as: complete regression (CR;non-palpable tumor) or partial regression (PR; 50% reduction in tumorvolume). Synergy is defined as antitumor activity significantly superior(p<0.05) to the activity of monotherapy with each agent.

In the SA1N fibrosarcoma tumor model, which is sensitive to PD-1blockade and modestly sensitive to (E1)-3s, interferon-α, and IMMU-132,borderline synergy is evident with the combination of hPD-1-v5 mAb and(E1)-3s, whereas no effect is observed with interferon-α. IMMU-132monotherapy does not produce significant SA1N antitumor activity.However, combining IMMU-132 with hPD-1-v5 mAb results in synergy. In theM109 lung metastasis model and CT26 colon carcinoma model, synergy isdetected for hPD-1-v5 mAb combined with each of IMMU-132, (E1)-3s andinterferon-α.

In summary, addition of hPD-1-v5 mAb to interferon-α, IMMU-132, or(E1)-3s results in model-dependent synergistic activities. Synergy isobserved regardless of the immunogenicity of the tumor and only when atleast one of the therapies is active. All combination regimens arewell-tolerated and the combination therapies do not appear to inhibithPD-1-v5 mAb activity. Synergy is observed in tumors unresponsive tohPD-1-v5 mAb alone, suggesting that the other therapeutic agents mightinduce immunogenic cell death.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A murine, chimeric or humanized anti-PD-1antibody or antigen-binding fragment thereof comprising the heavy chainCDR sequences GFAFSSNDMS (SEQ ID NO:1), TISGGGINTYYPDSVKG (SEQ ID NO:2)and RSNYAWFAY (SEQ ID NO:3) and the light chain CDR sequencesRASESVDTYGISFMN (SEQ ID NO:4), PNQGS (SEQ ID NO:5) and QQSKEVPWT (SEQ IDNO:6).
 2. The anti-PD-1 antibody of claim 1, wherein the anti-PD-1antibody is a chimeric antibody comprising the light chain amino acidsequence SEQ ID NO:8 and the heavy chain amino acid sequence SEQ IDNO:8.
 3. The anti-PD-1 antibody of claim 1, wherein the anti-PD-1antibody is a humanized antibody comprising the light chain amino acidsequence SEQ ID NO:9 and the heavy chain amino acid sequence SEQ IDNO:10.
 4. The anti-PD-1 antibody of claim 1, wherein the antibody is anIgG1, IgG2, IgG3 or IgG4 antibody.
 5. The anti-PD-1 antibody of claim 1,wherein the antibody has a G1m3 heavy chain and Km3 light chainallotype.
 6. The anti-PD-1 antibody of claim 1, wherein the antibodyfragment is selected from the group consisting of F(ab′)₂, F(ab)₂, Fab',Fab, Fv, scFv and dAb antibody fragments.
 7. The anti-PD-1 antibody ofclaim 1, wherein the antibody or antigen-binding fragment thereof is anaked antibody or antigen-binding fragment thereof
 8. A method oftreating a human cancer comprising administering to a human subject withcancer an anti-PD-1 antibody or antigen-binding fragment thereofcomprising the heavy chain CDR sequences GFAFSSNDMS (SEQ ID NO:1),TISGGGINTYYPDSVKG (SEQ ID NO:2) and RSNYAWFAY (SEQ ID NO:3) and thelight chain CDR sequences RASESVDTYGISFMN (SEQ ID NO:4), PNQGS (SEQ IDNO:5) and QQSKEVPWT (SEQ ID NO:6).
 9. The method of claim 8, wherein theanti-PD-1 antibody is a chimeric antibody comprising the light chainamino acid sequence SEQ ID NO:8 and the heavy chain amino acid sequenceSEQ ID NO:8.
 10. The method of claim 8, wherein the anti-PD-1 antibodyis a humanized antibody comprising the light chain amino acid sequenceSEQ ID NO:9 and the heavy chain amino acid sequence SEQ ID NO:10. 11.The method of claim 8, wherein the antibody or antigen-binding fragmentthereof is a naked antibody or antigen-binding fragment thereof.
 12. Themethod of claim 11, further comprising administering to the subject atherapeutic agent selected from the group consisting of a secondantibody, a second antigen-binding antibody fragment, an antibody-drugconjugate (ADC), a drug, a toxin, an enzyme, a cytotoxic agent, ananti-angiogenic agent, a pro-apoptotic agent, an antibiotic, a hormone,an immunomodulator, a cytokine, a chemokine, an interferon, an antisenseoligonucleotide, a small interfering RNA (siRNA), and a radioisotope.13. The method of claim 12, wherein the drug is selected from the groupconsisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogenreceptor binding agents, etoposide (VP16), etoposide glucuronide,etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, Pro-2-P-Dox, PSI-341, raloxifene,semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen,temazolomide, transplatinum, thalidomide, thioguanine, thiotepa,teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 14. The method ofclaim 12, wherein the chemokine is selected from the group consisting ofRANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.
 15. The method of claim12, wherein the immunomodulator is selected from the group consisting ofa cytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN), anderythropoietin.
 16. The method of claim 27, wherein the cytokine isselected from the group consisting of hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, tumor necrosis factor-α, tumor necrosis factor-β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), a nerve growth factor (NGF), NGF-β,platelet-growth factor, a transforming growth factors (TGF), TGF-α,TGF-β, insulin-like growth factor-I, insulin-like growth factor-II,erythropoietin (EPO), an osteoinductive factor, an interferon,interferon-α, interferon-β, interferon-λ, a colony stimulating factors(CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF),granulocyte-CSF (G-CSF), interleukin-1 (IL-1), IL-la, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin,thrombospondin, endostatin, tumor necrosis factor and LT (lymphotoxin).17. The method of claim 12, wherein the second antibody is a checkpointinhibitor antibody selected from the group consisting of pembrolizumab(MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224,MDX-1105, MEDI4736, atezolizumab (MPDL3280A), BMS-936559, ipilimumab,lirlumab, IPH2101, durvalumab and tremelimumab.
 18. The method of claim12, wherein the second antibody binds to an antigen selected from thegroup consisting of CTLA-4, PD-1, PD-L1, LAG3, B7-H3, B7-H4. KIR andTIM3.
 19. The method of claim 12, wherein the second antibody binds toan antigen selected from the group consisting of alpha-fetoprotein(AFP), a4 integrin, B7, carbonic anhydrase IX, complement factors C1q,C1r, C1s, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5a, C5aR, C5b, C5, C6,C7, C8, C9n, CCL19, CCL21, CD1, CD1a, CD2, CD3R, CD4, CD5, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD79b, CD80, CD83,CD86, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM-5, CEACAM-6, CSAp,DLL3, DLL4, ED-B of fibronectin, EGFR, EGP-1 (Trop-2), EGP-2, ErbB2,Factor H, FHL-1, fibrin, Flt-3, folate receptor, glycoprotein IIb/IIIa,gp41, gp120, GRO-β, HLA-DR, HM1.24, HM1.24, HMGB-1, hypoxia induciblefactor (HIF), Ia, ICAM-1, IFN-α, IFN-β, IFN-γ, IFN-λ, IgE, IGF-1R, IL-1,IL-1Ra, IL-2, IL-4R, IL-6, IL-6R, IL-8, IL-13R, IL-15R, IL-15, IL-17,IL-17R, IL-18, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,insulin-like growth factor-1 (ILGF-1), IP-10, KIR, Le(y),lipopolysaccharide (LPS), MAGE, MCP-1, mCRP, mesothelin, MIF, MIP-1A,MIP-1B, MUC1, MUC2, MUC3, MUC4, MUC5ac, NCA-90, NCA-95, NF-κB, PlGF,PSMA, RANTES, T101, TAC, TAG-72, tenascin, Thomson-Friedenreichantigens, thrombin, tissue factor, Tn antigen, TNF-α, TRAIL receptor (R1and R2), tumor necrosis antigens, VEGF, VEGFR and an oncogene product.20. The method of claim 12, wherein the second antibody is selected fromthe group consisting of hA19 (anti-CD19), hR1 (anti-IGF-1R), hPAM4(anti-MUC5ac), hA20 (anti-CD20), hIMMU31 (anti-AFP), hLL1 (anti-CD74),hLL2 (anti-CD22), hMu09 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14(anti-CEACAM5), hMN-15 (anti-CEACAM6), hMN-3 (anti-CEACAM6), and hRS7(anti-Trop-2).
 21. The method of claim 12, wherein the ADC is selectedfrom the group consisting of hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38,hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox,hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox,hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumabozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumabozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062,SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME,ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593,RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853,IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine.
 22. Themethod of claim 12, wherein the ADC is hRS7-SN-38 (IMMU-132),hMN-14-SN-38 (IMMU-130) or hL243-SN-38 (IMMU-140).
 23. The method ofclaim 12, wherein the interferon is selected from the group consistingof interferon-α, interferon-β, interferon-λ1, interferon-λ2 andinterferon-λ3.
 24. The method of claim 12, wherein the interferon isinterferon-α.
 25. The method of claim 12, wherein an ADC is administeredprior to any other agent.
 26. The method of claim 12, wherein theinterferon is administered as free interferon, PEGylated interferon, aninterferon fusion protein or interferon conjugated to an antibody. 27.The method of claim 8, wherein the cancer expresses PD-1.
 28. The methodof claim 8, wherein the cancer is selected from the group consisting ofacute lymphoblastic leukemia, acute myelogenous leukemia, biliarycancer, B-cell leukemia, B-cell lymphoma, biliary cancer, bone cancer,brain cancer, breast cancer, triple negative breast cancer, cervicalcancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colorectal cancer, endometrial cancer, esophagealcancer, gall bladder cancer, gastric cancer, gastrointestinal tractcancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin'slymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma,multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreaticcancer, prostate cancer, pulmonary tract cancer, renal cancer, sarcoma,skin cancer, testicular cancer, urothelial cancer, and urinary bladdercancer.
 29. A composition comprising an anti-PD-1 antibody according toclaim
 1. 30. A kit comprising: a) an anti-PD-1 antibody according toclaim 1; and b) at least one container.